The pre-membrane (pre-M) structural glycoprotein of dengue virus is cleaved during virus maturation to yield a membrane (M) protein, that becomes part of the virion, leaving a residual glycoprotein segment (Mresid). Vaccinia virus recombinants expressing these three proteins of dengue 4 (D4) virus were constructed. Cells infected with the recombinant expressing pre-M (vD4pM) produced two different forms of the pre-M protein. One form was apparently authentic pre-M that could be immunoprecipitated by polyvalent antiserum to dengue 4 virus as well as antiserum raised against an N-terminal peptide segment, and the other form was a comigrating protein, detectable only by antipeptide sera, that was linked into oligomeric forms through disulfide bonds. Similarly, cells infected with vD4 Mresid produced two forms of the same protein. These cells secreted the Mresid glycoprotein into the medium; this protein co-migrated with that secreted by D4 virus infected cells. The protein product of vD4 M was not detected by immunoprecipitation. Following immunization almost all mice inoculated with vD4 pM or vD4 M survived subsequent intracerebral challenge with 100 LD50 of homologous D4 virus, while control mice and mice previously inoculated with vD4 Mresid were not protected. Serum transfer experiments provided evidence that protection was mediated by antibodies. The role of pre-M as a protective antigen was further investigated by constructing vaccinia recombinants expressing dengue 2 structural proteins. A recombinant that expressed both D2 pre-M and the envelope (E) glycoproteins induced solid resistance to a 100 LD50 challenge with D2 virus, but recombinants which expressed E or pre-M alone were only partially protective. Protection was enhanced when mice were immunized with both vD2 pM and vD2 E. Thus, M constitutes a third, previously unrecognized, protective antigen of dengue virus that must be taken into consideration during the development of dengue virus vaccines.